Retinal pigmented epithelium and photoreceptor dual cell aggregates and methods of use thereof

Abstract

Provided herein is a dual cell aggregate culture of retinal epithelial cells and photoreceptors for use as a research tool, such as the screening of compounds or model for study of retinal diseases, as well as a therapeutic for treating ocular diseases.

What is claimed is: 
     
         1 . A dual cell aggregate composition comprising retinal pigment epithelium cells (RPE) with photoreceptors and/or photoreceptor precursor cells (PR/PRP). 
     
     
         2 . The composition of  claim 1 , wherein the composition is xeno-free, feeder-free, and defined. 
     
     
         3 . The composition of  claim 1  or  2 , wherein the RPE are mature RPE expressing Bestrophin-1 (BEST1) and/or ZO-1. 
     
     
         4 . The composition of  claim 1  or  2 , wherein the RPE are immature RPE with essentially no expression of BEST1 and/or ZO-1. 
     
     
         5 . The composition of any of  claims 1 - 4 , wherein the RPE are polarized. 
     
     
         6 . The composition of any of  claims 1 - 4 , wherein the RPE are not polarized. 
     
     
         7 . The composition of any of  claims 1 - 5 , wherein the composition is essentially free of a bioabsorbable scaffold and/or extracellular matrix (ECM) proteins. 
     
     
         8 . The composition of any of  claims 1 - 7 , wherein the ratio of PR/PRP to RPE is about 2:1 to about 500:1 at the time of assembly of the dual cell aggregate composition. 
     
     
         9 . The composition of any of  claims 1 - 7 , wherein the ratio of PR/PRP to RPE is about 1:1 to about 100:1 at the time of assembly of the dual cell aggregate composition. 
     
     
         10 . The composition of any of  claims 1 - 9 , wherein the RPE and/or the PR/PRP are derived from pluripotent stem cells (PSCs). 
     
     
         11 . The composition of  claim 10 , wherein PSCs are induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). 
     
     
         12 . The composition of  claim 11 , wherein the iPSCs are human iPSCs (hiPSCs). 
     
     
         13 . The composition of any of  claims 1 - 12 , wherein PR/PRP were not derived from organoids. 
     
     
         14 . The composition of any of  claims 1 - 13 , wherein the RPE and/or the PR/PRP have been previously cryopreserved. 
     
     
         15 . The composition of  claim 14 , wherein the cryopreserved RPE and/or PR/PRP have been thawed and cultured for at least one week. 
     
     
         16 . The composition of any of  claims 1 - 15 , wherein the RPE and PR/PRP are formed at a density of about 1 million cells/mL to about 10 million cells/mL. 
     
     
         17 . The composition of any of  claims 1 - 16 , wherein the RPE and PR/PRP are formed at a density of about 5 million cells/mL. 
     
     
         18 . The composition of any of  claims 1 - 18 , wherein the RPE and/or the PR/PRP are from same donor. 
     
     
         19 . The composition of any of  claims 1 - 18 , wherein the PR/PRP are rod-predisposed. 
     
     
         20 . The composition of any of  claims 1 - 18 , wherein the PR/PRP are cone-predisposed. 
     
     
         21 . A pharmaceutical composition comprising the dual cell aggregate composition of any of  claims 1 - 20 . 
     
     
         22 . The pharmaceutical composition of  claim 21 , further comprising hyaluronate. 
     
     
         23 . The pharmaceutical composition of  claim 22 , wherein the hyaluronate is added at a concentration of less than about 0.5%. 
     
     
         24 . The pharmaceutical composition of any of  claims 21 - 23 , further comprising sodium bicarbonate, calcium chloride, potassium chloride, potassium phosphate monobasic, magnesium chloride, magnesium sulfate, sodium chloride, and/or sodium phosphate dibasic. 
     
     
         25 . The pharmaceutical composition of any of  claims 21 - 24 , wherein the dual cell aggregate composition is cryopreserved. 
     
     
         26 . The pharmaceutical composition of any of  claims 21 - 25 , wherein the dual cell aggregate composition comprises 200,000 to 3,000,000 cells. 
     
     
         27 . The pharmaceutical composition of any of  claims 21 - 25 , wherein the dual cell aggregate composition comprises about 700,000 cells. 
     
     
         28 . A method for producing the dual cell aggregate composition of any of  claims 1 - 24  comprising seeding RPE and PR/PRP in culture medium comprising a ROCK inhibitor and culturing for a period of time sufficient to produce the dual cell aggregate composition. 
     
     
         29 . The method of  claim 28 , wherein the RPE and PR/PRP are seeded as essentially single-cell suspensions. 
     
     
         30 . The method of  claim 28 , wherein the RPE are seeded as an essentially single-cell suspension and the PR/PRP are seeded as aggregates. 
     
     
         31 . The method of any of  claims 28 - 30 , wherein the ROCK inhibitor is Y-27632. 
     
     
         32 . The method of  claim 31 , wherein the Y-27632 is added at a concentration of 10 μM. 
     
     
         33 . The method of any of  claims 28 - 32 , wherein the ratio of PR/PRP to RPE is about 2:1 to about 500:1 at the time of assembly of the dual cell aggregate composition. 
     
     
         34 . The method of any of  claims 28 - 32 , wherein the ratio of PR/PRP to RPE is about 100:1 at the time of assembly of the dual cell aggregate composition. 
     
     
         35 . The method of any of  claims 28 - 34 , wherein the culture medium further comprises prostaglandin E2 (PGE-2). 
     
     
         36 . The method of any of  claims 28 - 34 , wherein the RPE were previously cultured in the presence of PGE-2. 
     
     
         37 . The method of any of  claims 28 - 36 , wherein the PR/PRP express PRPH2. 
     
     
         38 . The method of any of  claims 28 - 37 , wherein the PR/PRP are rod-disposed. 
     
     
         39 . The method of any of  claims 28 - 38 , wherein the PR/PRP are cone-disposed. 
     
     
         40 . The method of any of  claims 28 - 39 , wherein the RPE and PR/PRP are seeded at a density of about 1 million cells/mL to about 10 million cells/mL. 
     
     
         41 . The method of any of  claims 28 - 40 , wherein the RPE and PR/PRP are seeded at a density of about 5 million cells/mL 
     
     
         42 . The method of any of  claims 28 - 41 , wherein the RPE and/or the PR/PRP have been previously cryopreserved. 
     
     
         43 . The method of any of  claims 28 - 42 , wherein the culture medium further comprises taurine and hydrocortisone. 
     
     
         44 . The method of any of  claims 28 - 43 , wherein the culture medium further comprises triiodothyronine. 
     
     
         45 . The method of any of  claims 28 - 44 , wherein the culture medium is defined media or serum-free media. 
     
     
         46 . The method of any of  claims 28 - 45 , wherein the culture medium comprises serum replacement. 
     
     
         47 . The method of any of  claims 28 - 46 , wherein culture medium is RPE-MM media. 
     
     
         48 . The method of any of  claims 28 - 47 , wherein the culturing is for at least 10 days. 
     
     
         49 . The method of any of  claims 28 - 47 , wherein the culturing is for 2 weeks to one month. 
     
     
         50 . The method of any of  claims 28 - 47 , wherein the culturing is for at least two months. 
     
     
         51 . The method of any of  claims 28 - 50 , wherein the culture medium is exchanged at least once every five days. 
     
     
         52 . The method of any of  claims 28 - 50 , wherein the culture medium is exchanged at least once every three days. 
     
     
         53 . The method of any of  claims 28 - 50 , wherein culture medium is exchanged at least once every other day. 
     
     
         54 . The method of any of  claims 28 - 53 , further comprising cryopreserving the dual cell aggregate composition. 
     
     
         55 . A method for treating an ocular injury or disorder in a subject comprising transplanting an effective amount of a dual cell aggregate composition of any of  claims 1 - 27  to an eye of the subject. 
     
     
         56 . The method of  claim 55 , wherein the subject is administered a dose of 200,000 to 3,000,000 of the RPE and PR/PRP cells. 
     
     
         57 . The method of  claim 55 , wherein the subject is administered a dose of about 700,000 of the RPE and PR/PRP cells. 
     
     
         58 . The method of  claim 55 , wherein the composition is transplanted to the subretinal space of the eye. 
     
     
         59 . The method of  claim 55  or  58 , wherein the ocular disorder is due to RPE dysfunction or photoreceptor dysfunction. 
     
     
         60 . The method of any of  claims 55 - 59 , wherein the ocular disorder is age-related macular degeneration, retinitis pigmentosa, cone-rod dystrophies, or Leber congenital amaurosis. 
     
     
         61 . The method of any of  claims 55 - 60 , wherein the dual cell aggregate composition produces both rod and cone photoreceptors in the eye of the subject. 
     
     
         62 . Use of the dual cell aggregate composition of any of  claims 1 - 20  as a model retina. 
     
     
         63 . A method of screening a compound comprising contacting one or more candidate compounds with the dual cell aggregate composition of any of  claims 1 - 20  and detecting an effect on the RPE-PRP dual cell aggregates. 
     
     
         64 . The method of  claim 63 , wherein the one or more candidate compounds are selected from the group consisting of a chemical compound, small molecule, polypeptide, growth factor, solvent, oligonucleotide, and cytokine. 
     
     
         65 . The method of  claim 63 , wherein detecting an effect comprises measuring cell proliferation, cell viability, cell death, drug toxicity, or retinal tissue maintenance or repair. 
     
     
         66 . The method of any of  claims 63 - 65 , wherein the method is in vivo. 
     
     
         67 . The method of any of  claims 63 - 65 , wherein the method is in vitro. 
     
     
         68 . The method of any of  claims 63 - 67 , wherein the method if performed in a high-throughput manner. 
     
     
         69 . The method of  claim 68 , wherein the dual cell aggregate composition is placed in a multi-well culture plate. 
     
     
         70 . An in vitro retina model comprising the dual cell aggregate composition of any of  claims 1 - 20 . 
     
     
         71 . The model of  claim 70 , wherein the RPE and/or PR/PRP are obtained from a disease cell line. 
     
     
         72 . The model of  claim 71 , wherein the disease is an ocular disease. 
     
     
         73 . The model of  claim 72 , wherein the ocular disease is age-related macular degeneration, retinitis pigmentosa, cone-rod dystrophies, or Leber congenital amaurosis.